PROJECT SUMMARY/ABSTRACT Foxp3+ CD4+ regulatory T (Treg) cells are critical for the prevention of autoimmunity and the regulation of antitumor immunity, functioning in part by suppressing the activation and function of conventional T cells. Treg cells depend on T cell receptor (TCR) stimulation for many facets of their biology, including thymic development, peripheral differentiation, and suppressive function. In this regard, the affinity of TCR binding to self-peptides complexed with MHC class II molecules (self-pMHCII) is thought to be a primary determinant of Treg cell development and function. Existing models of thymic selection posit that intermediate TCR-pMHCII affinities ? at the threshold between positive and negative selection ? promote optimal thymic Treg (tTreg) development, allowing Treg cell clones to differentiate while also evading clonal deletion. In addition, it has been suggested that Treg cells may be more sensitive to self-pMHC-II ligand than Foxp3neg conventional CD4+ T (Tconv) cells of the same specificity, potentially contributing to Treg-mediated suppression in the periphery. However, these proposed principles largely stem from the study of CD4+ T cells reactive to transgene-expressed foreign model antigens. As the expression patterns, ligand densities, and TCR-pMHCII affinities of such engineered antigen systems may not reflect those of endogenous self-antigens, it remains unclear whether the predictions of the affinity model apply to Treg cell clones reactive to natural self-ligands. To address this question, we have identified a panel of CD4+ T cell clones exhibiting varying sensitivities to the same endogenous tissue-restricted antigen (TRA), Tcaf3646-658 (?C4? peptide). C4 is an unmodified I-Ab restricted prostatic self-peptide that drives the thymic differentiation of a canonical Treg cell clone, MJ23. In other work, we also demonstrated that monoclonal MJ23 Treg cells are highly enriched in oncogene-driven prostate tumors, indicating that the C4 peptide is a naturally occurring tumor-associated antigen. Our new data using C4/I-Ab tetramers demonstrate that, in prostate-tumor-bearing mice, C4-reactive Treg and Tconv cells coexist in the tumor-draining lymph nodes, yet only the former is detected within the tumor infiltrate. Whether TCR-pMHCII affinity contributes to Tconv cell exclusion from prostate tumors remains unclear. Thus, this proposal seeks to define the role of TCR- pMHCII affinity in the thymic and peripheral selection of CD4+ T cells reactive to the natural TRA and tumor- associated antigen C4. The studies in Aim 1 will provide the first evidence testing the TCR affinity model for T cell clones reactive to a natural self-peptide that directs tTreg cell differentiation, offering a unique opportunity to validate and/or upend existing paradigms of thymic selection. The studies in Aim 2 mice will yield unique mechanistic insights into the factors driving the peripheral selection of TRA-reactive Treg and Tconv cells, and the selective enrichment of the former into the tumor microenvironment. Collectively, this work will leverage a novel and physiologically relevant TCR-ligand system to understand the impact of TCR-pMHCII affinity on the fundamental mechanisms by which immune tolerance is established and enforced.